Mechanisms of nucleation and stabilization in clathrin-mediated endocytosis Clathrin-mediated endocytosis (CME) is the main mechanism by which cells internalize molecules from the extracellular space. As such, it is involved in all functions regulating cellular homeostasis. CME can occur constitutively or can be stimulated by ligand-bound receptors. CME begins with the assembly of clathrin-coated pits (CCPs) at the membrane which stabilize, mature, invaginate, and pinch-off as sealed clathrin-coated vesicles (CCVs). During maturation the pits accumulate cargo, adaptors, accessory proteins, and clathrin. An emerging theme in the field of mammalian endocytosis is the notion that endocytic pits vary in molecular composition, lifetime, size, and even on whether they form CCVs or not. We do not yet understand what is the source or the functional significance of this heterogeneity in CME. While it is likely that the observed heterogeneity could result from many different sources, it is probable that the variability reflects the intrinsic heterogeneity in the regulation of CME. Data from the Danuser and Schmid laboratories suggest that most of the regulation of CME occurs during the difficult to study early stages of an endocytic pit's lifetime. In particular, it is probable that a large part of the observed heterogeneity is imparted during nucleation. The goal of this research is to establish the mechanisms which regulate the nucleation of CCPs, and to determine the sources and effects of heterogeneity during these initiation events. I propose to begin by studying the spatial regulation of nucleation as a possible source of heterogeneity in CME. The concept of stable nucleation machinery that creates multiple nucleations in one spot, called the hot-spot, has been previously suggested, but is a source of debate in the field. I propose to resolve whether the heterogeneity of CCPs can be explained in part by their spatial organization at the plasma membrane. I propose to identify the proteins that spatially regulate nucleation. I propose to look at the residence time of various nucleation resources at sites of repeated nucleation to determine if a stable nucleation structure exists. Another possible source of heterogeneity and regulation during the nucleation and stabilization phases of CME is the composition of pits at these early stages. I propose to measure the stoichiometry of the proteins that form the nucleating pit. PUBLIC HEALTH RELEVANCE: Clathrin-mediated endocytosis (CME) is the main mechanism by which cells internalize molecules from the extracellular space. As such, it is involved in all functions regulating cellular homeostasis. Deregulation of CME is therefore relevant in many diseases.